1-pyrazolyl-3- (4- ((2 -anilinopyrimidin- 4 - yl) oxy) napththalen- i - yl) ureas as p38 mapkinase inhibitors

ABSTRACT

There is provided a compound of formula (I) which is an inhibitor of the family of p38 mitogen-activated protein kinase enzymes, and to its use in therapy, including in pharmaceutical combinations, especially in the treatment of inflammatory diseases, including inflammatory diseases of the lung, such as asthma and COPD.

FIELD OF THE INVENTION

The invention relates to compounds which are inhibitors of the family ofp38 mitogen-activated protein kinase enzymes (referred to herein as p38MAP kinase inhibitors), for example the alpha and gamma kinase sub-typesthereof, and of Syk kinase and the Src family of tyrosine kinases, andto their use in therapy, including in pharmaceutical combinations,especially in the treatment of inflammatory diseases, in particularinflammatory diseases of the lung, such as asthma and COPD, as well asthose of the gastrointestinal tract, such as ulcerative colitis andCrohn's disease and of the eye, such as uveitis.

BACKGROUND OF THE INVENTION

Four p38 MAPK isoforms (alpha, beta, gamma and delta respectively), havebeen identified each displaying different patterns of tissue expressionin man. The p38 MAPK alpha and beta isoforms are found ubiquitously inthe body, being present in many different cell types. The alpha isoformis well characterized in terms of its role in inflammation. Althoughstudies using a chemical genetic approach in mice indicate that the p38MAPK beta isoform does not play a role in inflammation (O'Keefe, S. J.et al., J. Biol. Chem., 2007, 282(48):34663-71.), it may be involved inpain mechanisms through the regulation of COX2 expression (Fitzsimmons,B. L. et al., Neuroreport, 2010, 21(4):313-7). These isoforms areinhibited by a number of previously described small molecular weightcompounds. Early classes of inhibitors were highly toxic due to thebroad tissue distribution of these isoforms which resulted in multipleoff-target effects of the compounds. Furthermore, development of asubstantial number of inhibitors has been discontinued due tounacceptable safety profiles in clinical studies (Pettus, L. H. andWurz, R. P., Curr. Top. Med. Chem., 2008, 8(16):1452-67.). As theseadverse effects vary with chemotype, and the compounds have distinctkinase selectivity patterns, the observed toxicities may bestructure-related rather than p38 mechanism-based.

Less is known about the p38 MAPK gamma and delta isoforms, which, unlikethe alpha and beta isozymes are expressed in specific tissues and cells.The p38 MAPK-delta isoform is expressed more highly in the pancreas,testes, lung, small intestine and the kidney. It is also abundant inmacrophages and detectable in neutrophils, CD4+ T cells and inendothelial cells (Shmueli, O. et al., Comptes Rendus Biologies, 2003,326(10-11)1067-1072; Smith, S. J. Br. J. Pharmacol., 2006, 149:393-404;Hale, K. K., J. Immunol., 1999, 162(7):4246-52; Wang, X. S. et al., J.Biol. Chem., 1997, 272(38):23668-23674.) Very little is known about thedistribution of p38 MAPK gamma although it is expressed more highly inbrain, skeletal muscle and heart, as well as in lymphocytes andmacrophages (Shmueli, O. et al., Comptes Rendus Biologies, 2003,326(10-11):1067-1072; Hale, K. K., J. Immunol., 1999, 162(7):4246-52;Court, N. W. et al., J. Mol. Cell. Cardiol., 2002, 34(4):413-26;Mertens, S. et al., FEBS Lett., 1996, 383(3):273-6.).

Selective small molecule inhibitors of p38 MAPK gamma and p38 MAPK deltaare not currently available, although one previously disclosed compound,BIRB 796, is known to possess pan-isoform inhibitory activity. Theinhibition of p38 MAPK gamma and delta isoforms is observed at higherconcentrations of the compound than those required to inhibit p38 MAPKalpha and p38 beta (Kuma, Y., J. Biol. Chem., 2005, 280:19472-19479.).In addition BIRB 796 also impaired the phosphorylation of p38 MAPKs orJNKs by the upstream kinase MKK6 or MKK4. Kuma discussed the possibilitythat the conformational change caused by the binding of the inhibitor tothe MAPK protein may affect the structure of both its phosphorylationsite and the docking site for the upstream activator, thereby impairingthe phosphorylation of p38 MAPKs or JNKs.

p38 MAP kinase is believed to play a pivotal role in many of thesignalling pathways that are involved in initiating and maintainingchronic, persistent inflammation in human disease, for example, insevere asthma and in COPD (Chung, F., Chest, 2011, 139(6):1470-1479.).There is now an abundant literature which demonstrates that p38 MAPkinase is activated by a range of pro-inflammatory cytokines and thatits activation results in the recruitment and release of additionalpro-inflammatory cytokines. Indeed, data from some clinical studiesdemonstrate beneficial changes in disease activity in patients duringtreatment with p38 MAP kinase inhibitors. For instance Smith describesthe inhibitory effect of p38 MAP kinase inhibitors on TNFα (but notIL-8) release from human PBMCs.

The use of inhibitors of p38 MAP kinase in the treatment of chronicobstructive pulmonary disease (COPD) has also been proposed. Smallmolecule inhibitors targeted to p38 MAPK a/13 have proved to beeffective in reducing various parameters of inflammation in cells and intissues obtained from patients with COPD, who are generallycorticosteroid insensitive, (Smith, S. J., Br. J. Pharmacol., 2006,149:393-404.) as well as in various in vivo animal models (Underwood, D.C. et al., Am. J. Physiol., 2000, 279:L895-902; Nath, P. et al., Eur. J.Pharmacol., 2006, 544:160-167.). Irusen and colleagues have alsosuggested the possible involvement of p38 MAPK a/13 with corticosteroidinsensitivity via the reduction of binding affinity of theglucocorticoid receptor (GR) in nuclei (Irusen, E. et al., J. AllergyClin. Immunol., 2002, 109:649-657.). Clinical experience with a range ofp38 MAP kinase inhibitors, including AMG548, BIRB 796, VX702, SCI0469and SCI0323 has been described (Lee, M. R. and Dominguez, C., CurrentMed. Chem., 2005, 12:2979-2994.).

COPD is a condition in which the underlying inflammation is reported tobe substantially resistant to the anti-inflammatory effects of inhaledcorticosteroids. Consequently, a superior strategy for treating COPDwould be to develop an intervention which has both inherentanti-inflammatory effects and the ability to increase the sensitivity ofthe lung tissues of COPD patients to inhaled corticosteroids. A recentpublication of Mercado (Mercado, N., et al., Mol. Pharmacol., 2011,80(6):1128-1135.) demonstrates that silencing p38 MAPK γ has thepotential to restore sensitivity to corticosteroids. Consequently theremay be a dual benefit for patients in the use of a p38 MAP kinaseinhibitor for the treatment of COPD and severe asthma. However, themajor obstacle hindering the utility of p38 MAP kinase inhibitors in thetreatment of human chronic inflammatory diseases has been the severetoxicity observed in patients resulting in the withdrawal from clinicaldevelopment of many compounds including all those specifically mentionedabove.

Many patients diagnosed with asthma or with COPD continue to suffer fromuncontrolled symptoms and from exacerbations of their medical conditionthat can result in hospitalisation. This occurs despite the use of themost advanced, currently available treatment regimens, comprising ofcombination products of an inhaled corticosteroid and a long actingβ-agonist. Data accumulated over the last decade indicates that afailure to manage effectively the underlying inflammatory component ofthe disease in the lung is the most likely reason that exacerbationsoccur. Given the established efficacy of corticosteroids asanti-inflammatory agents and, in particular, of inhaled corticosteroidsin the treatment of asthma, these findings have provoked intenseinvestigation. Resulting studies have identified that some environmentalinsults invoke corticosteroid-insensitive inflammatory changes inpatients' lungs. An example is the response arising fromvirally-mediated upper respiratory tract infections (URTI), which haveparticular significance in increasing morbidity associated with asthmaand COPD.

Epidemiological investigations have revealed a strong associationbetween viral infections of the upper respiratory tract and asubstantial percentage of the exacerbations suffered by patients alreadydiagnosed with chronic respiratory diseases. Some of the most compellingdata in this regard derives from longitudinal studies of childrensuffering from asthma (Papadopoulos, N. G., Papi, A., Psarras, S. andJohnston, S. L., Paediatr. Respir. Rev. 2004, 5(3):255-260.). A varietyof additional studies support the conclusion that a viral infection canprecipitate exacerbations and increase disease severity. For example,experimental clinical infections with rhinovirus have been reported tocause bronchial hyper-responsiveness to histamine in asthmatics that isunresponsive to treatment with corticosteroids (Grunberg, K., Sharon, R.F., et al., Am. J. Respir. Crit. Care Med., 2001, 164(10):1816-1822.).Further evidence derives from the association observed between diseaseexacerbations in patients with cystic fibrosis and HRV infections (Wat,D., Gelder, C., et al., J. Cyst. Fibros. 2008, 7:320-328.). Alsoconsistent with this body of data is the finding that respiratory viralinfections, including rhinovirus, represent an independent risk factorthat correlates negatively with the 12 month survival rate inpaediatric, lung transplant recipients (Liu, M., Worley, S., et al.,Transpi. Infect. Dis. 2009, 11(4):304-312.).

Clinical research indicates that the viral load is proportionate to theobserved symptoms and complications and, by implication, to the severityof inflammation. For example, following experimental rhinovirusinfection, lower respiratory tract symptoms and bronchialhyper-responsiveness correlated significantly with virus load (Message,S. D., Laza-Stanca, V., et al., PNAS, 2008; 105(36):13562-13567.).Similarly, in the absence of other viral agents, rhinovirus infectionswere commonly associated with lower respiratory tract infections andwheezing, when the viral load was high in immunocompetent paediatricpatients (Gerna, G., Piralla, A., et al., J. Med. Virol. 2009,81(8):1498-1507.).

Interestingly, it has been reported recently that prior exposure torhinovirus reduced the cytokine responses evoked by bacterial productsin human alveolar macrophages (Oliver, B. G., Lim, S., et al., Thorax,2008, 63:519-525.). Additionally, infection of nasal epithelial cellswith rhinovirus has been documented to promote the adhesion of bacteria,including S. aureus and H. influenzae (Wang, J. H., Kwon, H. J. andYong, J. J., The Laryngoscope, 2009, 119(7):1406-1411.). Such cellulareffects may contribute to the increased probability of patientssuffering a lower respiratory tract infection following an infection inthe upper respiratory tract. Accordingly, it is therapeutically relevantto focus on the ability of novel interventions to decrease viral load ina variety of in vitro systems, as a surrogate predictor of their benefitin a clinical setting.

High risk groups, for whom a rhinovirus infection in the upperrespiratory tract can lead to severe secondary complications, are notlimited to patients with chronic respiratory disease. They include, forexample, the immune compromised who are prone to lower respiratory tractinfection, as well as patients undergoing chemotherapy, who face acute,life-threatening fever. It has also been suggested that other chronicdiseases, such as diabetes, are associated with a compromisedimmuno-defence response. This increases both the likelihood of acquiringa respiratory tract infection and of being hospitalised as a result(Peleg, A. Y., Weerarathna, T., et al., Diabetes Metab. Res. Rev., 2007,23(1):3-13; Kornum, J. B., Reimar, W., et al., Diabetes Care, 2008,31(8):1541-1545.).

Whilst upper respiratory tract viral infections are a cause ofconsiderable morbidity and mortality in those patients with underlyingdisease or other risk factors; they also represent a significanthealthcare burden in the general population and are a major cause ofmissed days at school and lost time in the workplace (Rollinger, J. M.and Schmidtke, M., Med. Res. Rev., 2010, Doi 10.1002/med.20176.). Theseconsiderations make it clear that novel medicines, that possess improvedefficacy over current therapies, are urgently required to prevent andtreat rhinovirus-mediated upper respiratory tract infections. In generalthe strategies adopted for the discovery of improved antiviral agentshave targeted various proteins produced by the virus, as the point oftherapeutic intervention. However, the wide range of rhinovirusserotypes makes this a particularly challenging approach to pursue andmay explain why, at the present time, a medicine for the prophylaxis andtreatment of rhinovirus infections has yet to be approved by anyregulatory agency.

Viral entry into the host cell is associated with the activation of anumber of intracellular signalling pathways which are believed to play aprominent role in the initiation of inflammatory processes (reviewed byLudwig, S, 2007; Signal Transduction, 7:81-88.) and of viral propagationand subsequent release. One such mechanism, which has been determined toplay a role in influenza virus propagation in vitro, is activation ofthe phosphoinositide 3-kinase/Akt pathway. It has been reported thatthis signalling pathway is activated by the NS1 protein of the virus(Shin, Y. K., Liu, Q. et al., J. Gen. Virol., 2007, 88:13-18.) and thatits inhibition reduces the titres of progeny virus (Ehrhardt, C.,Marjuki, H. et al., Cell Microbiol., 2006, 8:1336-1348.).

Furthermore, the MEK inhibitor 00126 has been documented to inhibitviral propagation without eliciting the emergence of resistant variantsof the virus (Ludwig, S., Wolff, T. et al., FEBS Lett., 2004,561(1-3):37-43.). More recently, studies targeting inhibition of Sykkinase have demonstrated that the enzyme plays an important role inmediating rhinovirus entry into cells and also virus-inducedinflammatory responses, including ICAM-1 up-regulation (Sanderson, M.P., Lau, C. W. et al., Inflamm. Allergy Drug Targets, 2009, 8:87-95.).Syk activity is reported to be controlled by c-Src as an upstream kinasein HRV infection (Lau, C. et al., J. Immunol., 2008, 180(2):870-880.). Asmall number of studies have appeared that link the activation ofcellular Src (Src1 or p60-Src) or Src family kinases to infection withviruses. These include a report that adenovirus elicits a PI3 kinasemediated activation of Akt through a c-Src dependent mechanism. It hasalso been suggested that Rhinovirus-39 induced IL-8 production inepithelial cells depends upon Src kinase activation (Bentley, J. K.,Newcomb, D. C., J. Virol., 2007, 81:1186-1194.). Finally, it has beenproposed that activation of Src kinase is involved in the induction ofmucin production by rhinovirus-14 in epithelial cells and sub-mucosalglands (Inoue, D. and Yamaya, M., Respir. Physiol. Neurobiol., 2006,154(3):484-499.).

It has been disclosed previously that compounds that inhbit the activityof both c-Src and Syk kinases are effective agents against rhinovirusreplication (Charron, C. E. et al., WO 2011/158042.) and that compoundsthat inhibit p59-HCK are effective against influenza virus replication(Charron, C. E. et al., WO 2011/070369.). For the reasons summarisedabove, compounds designed to treat chronic respiratory diseases thatcombine these inherent properties with the inhibition of p38 MAPKs, areexpected to be particularly efficacious.

Certain p38 MAPK inhibitors have also been described as inhibitors ofthe replication of respiratory syncitial virus (Cass, L. et al., WO2011/158039.).

Furthermore, it is noteworthy that a p38 MAPK inhibitor was found todeliver benefit for patients with IBD after one week's treatment whichwas not sustained over a four week course of treatment (Schreiber, S. etal., Clin. Gastro. Hepatology, 2006, 4:325-334.).

In addition to playing key roles in cell signalling events which controlthe activity of pro-inflammatory pathways, kinase enzymes are now alsorecognised to regulate the activity of a range of cellular functions.Among those which have been discussed recently are the maintenance ofDNA integrity (Shilo, Y. Nature Reviews Cancer, 2003, 3:155-168.) andco-ordination of the complex processes of cell division. An illustrationof recent findings is a publication describing the impact of a set ofinhibitors acting upon the so-called “Olaharsky kinases” on thefrequency of micronucleus formation in vitro (Olaharsky, A. J. et al.,PLoS Comput. Biol., 2009, 5(7):e1000446.). Micronucleus formation isimplicated in, or associated with, disruption of mitotic processes andis therefore an undesirable manifestation of potential toxicity.Inhibition of glycogen synthase kinase 3α (GSK3α) was found to be aparticularly significant factor that increases the likelihood of akinase inhibitor promoting micronucleus formation. Recently, inhibitionof the kinase GSK3β with RNAi was also reported to promote micronucleusformation (Tighe, A. et al., BMC Cell Biology, 2007, 8:34.).

It may be possible to attenuate the adverse effects arising from druginteractions with Olaharsky kinases, such as GSK3α, by optimisation ofthe dose and/or by changing the route of administration. However, itwould be more advantageous to identify therapeutically useful moleculesthat demonstrate low or undectable activity against these off-targetenzymes and consequently elicit little or no disruption of mitoticprocesses, as measured in mitosis assays.

It is evident from consideration of the literature cited hereinabovethat there remains a need to identify and develop new p38 MAP kinaseinhibitors that have improved therapeutic potential over currentlyavailable treatments. Desirable compounds are those that exhibit asuperior therapeutic index by exerting, at the least, an equallyefficacious effect as previous agents but, in one or more respects, areless toxic at the relevant therapeutic dose. An objective of the presentinvention therefore, is to provide such novel compounds that inhibit theenzyme activity of p38 MAP kinase, for example with certain sub-typespecificities, together with Syk kinase and tyrosine kinases within theSrc family (particularly c-Src) thereby possessing goodanti-inflammatory properties, and suitable for use in therapy.

The Compound (I) exhibits a longer duration of action and/or persistenceof action in comparison to the previously disclosed allosteric p38 MAPkinase inhibitor BIRB 796 (Pargellis, C. et al., Nature Struct. Biol.,2002, 9(4):268-272.). An additional embodiment provides such novelcompound in one or more solid, crystalline forms that possess highchemical and physical stability suitable for formulation as inhaledmedicaments.

SUMMARY OF THE INVENTION

Thus in one aspect of the invention there is provided a compound offormula (I):

or a pharmaceutically acceptable salt or solvate thereof, including allstereoisomers and tautomers thereof.

“Compound of formula (I)” may also be referred to herein as “Compound(1)”.

In another aspect of the invention there is provided Compound (I) asdefined above as the free base.

In another aspect of the invention there is provided Compound (I) asdefined above as the anhydrous free base.

In another aspect of the invention there is provided Compound (I) asdefined above as the anhydrous free base in solid crystalline form.

In a further aspect of the invention there is provided Compound (I) asdefined above as the anhydrous free base in solid crystallinepolymorphic form A.

In a further aspect of the invention there is provided Compound (I) asdefined above as the anhydrous free base in solid crystallinepolymorphic form B.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an X-ray powder diffraction (XRPD) pattern obtained from asample of Compound (I) as the anhydrous free base in solid crystallinepolymorphic form A.

FIG. 2 displays an XRPD pattern acquired from a sample of Compound (I)as the anhydrous free base in solid crystalline polymorphic form B, postmicronization.

FIG. 3 reveals the results of thermogravimetric analysis of a sample ofCompound (I) as the anhydrous free base in solid crystalline polymorphicform B, post micronization.

FIG. 4 represents dynamic vapour sorption (DVS) isotherm plots derivedfrom samples of Compound (I) as the anhydrous free base in solidcrystalline polymorphic form B post micronization.

FIG. 5 represents the results of a hysterisis experiment conducted onCompound (I) as the anhydrous free base in solid, crystalline,polymorphic form B, post micronization, to determine the degree and rateof moisture absorption/desorption with time against changes in relativehumidity.

FIG. 6 is the infrared (IR) spectrum obtained from a sample of Compound(I) as the anhydrous free base in solid crystalline polymorphic form Bpost micronization.

FIG. 7 shows thermal analysis of a sample of Compound (I) as theanhydrous free base in solid crystalline polymorphic form B (micronized)by differential scanning calorimetry (DSC).

DETAILED DESCRIPTION OF THE INVENTION

The compound of formula (I) disclosed herein is:1-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(4-(2-(phenylamino)pyrimidin-4-yloxy)naphthalen-1-yl)urea.Examples of salts of Compound (I) include all pharmaceuticallyacceptable salts, such as, without limitation, acid addition salts ofstrong mineral acids such as HCl and HBr salts and addition salts ofstrong organic acids such as methanesulfonic acid.

As employed herein below the definition of a compound of formula (I) isintended to include salts, solvates, and all tautomers of said compound,unless the context specifically indicates otherwise. Examples ofsolvates include hydrates.

The invention provided herein extends to prodrugs of the compound offormula (I), that is to say compounds which break down and/or aremetabolised in vivo to provide an active compound of formula (I).General examples of prodrugs include simple esters, and other esterssuch as mixed carbonate esters, carbamates, glycosides, ethers, acetalsand ketals.

The invention embraces all isotopic derivatives of Compound (I). Thusthe invention embraces compounds which are compounds of Compound (I)having one or more atoms that have been replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numbermost commonly found in nature, or in which the proportion of an atomhaving an atomic mass or mass number found less commonly in nature hasbeen increased (the latter concept being referred to as “isotopicenrichment”). Thus the compounds of the disclosure include those wherethe atom specified is a naturally occurring or non-naturally occurringisotope. In one embodiment the isotope is a stable isotope. Thus thecompounds of the disclosure include, for example deuterium containingcompounds and the like. Thus, in one embodiment of the inventionCompound (I) contains an enriched level of deuterium in one or morehydrogen atoms (e.g. for a given hydrogen atom the level of thedeuterium isotope exceeds 20%, 50%, 75%, 90%, 95% or 99% by number).Examples of other isotopes that can be incorporated into Compound (I) orenriched in Compound (I) include isotopes of hydrogen, carbon, nitrogen,oxygen, fluorine, iodine and chlorine such as ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N,¹⁸F, ¹²³I or ¹²⁵I, which may be naturally occurring or non-naturallyoccurring isotopes.

In a further aspect of the invention there is provided one or moremetabolites of the compound of formula (I), in particular a metabolitethat retains one or more of the therapeutic activities of the compoundof formula (I). A metabolite, as employed herein, is a compound that isproduced in vivo from the metabolism of the compound of formula (I),such as, without limitation, oxidative metabolites and/or metabolitesgenerated, for example, from O-dealkylation.

The disclosure also extends to all polymorphic forms of the compoundsherein defined.

A route suitable for the preparation of the compound of formula (I) isshown below (Scheme 1).

Protective groups may be required to protect chemically sensitive groupsduring one or more of the reactions described above, to ensure that theprocess can be carried out and/or is efficient. Thus if desired ornecessary, intermediate compounds may be protected by the use ofconventional protective groups. Protective groups and the means fortheir removal are described in “Protective Groups in Organic Synthesis”,by Theodora W. Greene and Peter G. M. Wuts, published by John Wiley &Sons Inc; 4th Rev Ed., 2006, ISBN-10: 0471697540.

A detailed preparation of Compound (I) is provided in Example 1.

Novel intermediates as described herein form an aspect of the invention.

In another aspect of the invention, there is provided Compound (I) asthe anhydrous free base in solid, crystalline form. In a further aspectof the invention, there is provided Compound (I) as the anhydrous freebase in solid, crystalline, polymorphic form A which may be obtained,for example, by crystallising Compound (I) from isopropyl acetate. In aparticular aspect of the invention, there is provided Compound (I) asthe anhydrous free base in solid, crystalline, polymorphic form B, whichmay be obtained, for example, by crystallising Compound (I) from acetoneand water. A typical ratio of acetone to water that is suitable for thisprocess is between 5:1 and 200:1 e.g. around 10:1. Alternatively, form Bmay be obtained by crystallising Compound (I) from acetone alone.Detailed preparations of Compound (I) as the anhydrous free base insolid crystalline polymorphic forms A and B are provided in Examples 1and 3 of the Experimental Section, respectively.

In a further aspect of the invention, the solid state properties ofCompound (I) may be improved by further slurrying or recrystallizationsteps to produce, for example, material with improved morphology and/orcontaining a reduced level of residual solvent. For example, residualsolvent may be removed from Compound (I) as the anhydrous free base insolid, crystalline, polymorphic form B by slurrying Compound (I) inpolymorphic form B, in water, or alternatively by furtherrecrystallization from acetone. A detailed description of an exemplaryslurrying procedure is provided in Example 3a of the ExperimentalSection.

In one embodiment, there is provided solid, crystalline polymorphic formA of Compound (I) as the anhydrous free base having an XRPD patternsubstantially as shown in FIG. 1. The method of obtaining the XRPD datais described in Analytical Methods and the data discussed in Example 5.

Thus, there is provided Compound (I) as the anhydrous free base insolid, crystalline, polymorphic form A having an XRPD pattern with atleast one (for example one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen or all sixteen)peak(s) at 7.8, 8.7, 10.3, 11.2, 12.4, 15.2, 16.2, 17.5, 19.7, 20.8,22.6, 23.1, 24.6, 25.5, 26.7, 27.4 (±0.2 degrees, 2-theta values), thesepeaks being characteristic of the solid, crystalline, polymorphic formA. The peaks at 10.3, 15.2, 17.5, 23.1, 24.6, 26.7 and 27.4 areparticularly characteristic for the solid, crystalline, polymorphic formA and therefore it is preferable that at least one (for example one,two, three, four, five, six or all seven) of these peaks is observablein the XRPD pattern.

In another embodiment, there is provided solid, crystalline, polymorphicform B of Compound (I) as the anhydrous free base (micronized) having anXRPD pattern substantially as shown in FIG. 2. The method ofmicronization is described in Example 4 and the method of obtaining theXRPD data is described in Analytical Methods and the data discussed inExample 5.

Thus, there is provided Compound (I) as the anhydrous free base in solidcrystalline polymorphic form B (micronized) having an XRPD pattern withat least one (for example one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen or all eighteen) peaks at 3.9, 6.1, 7.7, 8.6, 10.9, 11.8,12.7, 14.3, 15.9, 16.7, 18.3, 18.7, 19.9, 20.9, 22.0, 22.6, 25.2, 28.9(±0.2 degrees, 2-theta values), these peaks being characteristic of thesolid crystalline polymorphic form B. The peaks at 3.9, 6.1, 11.8, 14.3,16.7, 18.3, 18.7 and 28.9 are particularly characteristic for the solid,crystalline, polymorphic form B and therefore it is preferable that atleast one (for example one, two, three, four, five, six, seven or alleight) of these peaks is observable in the XRPD pattern.

The melting points of Compound (I) as the anhydrous free base in solid,crystalline, polymorphic forms A and B were determined usingdifferential scanning calorimetry as described in Example 6. Compound(I) as the anhydrous free base in solid, crystalline, polymorphic form Awas found to have a melting point of 191.6° C., and Compound (I) as theanhydrous free base in solid, crystalline, polymorphic form B was foundto have a melting point of 214° C. Polymorphic form B was also found tohave a higher heat of fusion than polymorphic form A. As explained inExample 6, these results suggest that polymorphic form B isthermodynamically more stable than polymorphic form A.

The physical and chemical stabilities of Compound (I) as the anhydrousfree base in solid, crystalline, polymorphic form B, were investigated,the results of which are disclosed herein.

In order to assess physical stability, Compound (I) as the anhydrousfree base in solid, crystalline, polymorphic form B was micronizedfollowing the procedure described in Example 4, and samples of theresulting material were stored in open containers and subjected todifferent ambient temperatures and relative humidities. The physicalproperties and stabilities of the samples were investigated using TGA,DSC, DVS, IR spectroscopy and XRPD analysis. Full experimentalprocedures are provided in the General Procedures section and theresults are summarised in Example 7 (Table 7). As discussed in Example7, Compound (I) as the anhydrous free base in solid, crystalline,polymorphic form B (micronized) was found to have good physicalstability. The same experimental procedures were also carried out usingCompound (I) as the anhydrous free base in solid, crystalline,polymorphic form B in non-micronized form and the results were found tobe substantially similar to those obtained for the micronized materiali.e. Compound (I) as the anhydrous free base in solid, crystalline,polymorphic form B in both micronized and unmicronized forms was foundto have good physical stability.

In order to assess chemical stability, Compound (I) as the anhydrousfree base in solid, crystalline, polymorphic form B was micronizedfollowing the procedure described in Example 4. Micronized samples werestored in open containers and subjected to different ambienttemperatures and relative humidities. The chemical stabilities of thesamples were analysed by HPLC. The results are summarised in Example 8(Table 8) where it is indicated that Compound (I) as the anhydrous freebase in solid, crystalline, polymorphic form B, post microniastion wasfound to be chemically stable, although some sensitivity towards lightwas detected.

As a result of the solid state studies disclosed herein, it is concludedthat Compound (I) as the anhydrous free base in solid, crystalline,polymorphic form B can be micronized and that the resulting material hasgood physical and chemical stability.

The compound of formula (I) is a p38 MAP kinase inhibitor (especially ofthe alpha subtype) and in one aspect the compound is useful in thetreatment of inflammatory diseases, for example COPD and/or asthma.

Surprisingly, the compound exhibits a long duration of action and/orpersistence of action in comparison to the previously disclosed p38 MAPkinase inhibitor BIRB796.

Persistence of action as used herein is related to the dissociation rateor dissociation constant of the compound from the target (such as areceptor). A low dissociation rate may lead to persistence.

A low dissociation rate in combination with a high association ratetends to provide potent therapeutic entities.

The compound of formula (I) is expected to be potent in vivo.

Typically, the prior art compounds developed to date have been intendedfor oral administration. This strategy involves optimizing compoundswhich achieve their duration of action by an appropriate pharmacokineticprofile, thereby ensuring that a sufficiently high drug concentration isestablished and maintained between doses to provide clinical benefit.The inevitable consequence of this approach is that all bodily tissues,and especially the liver and the gut, are exposed tosupra-therapeutically active concentrations of the drug, whether or notthey are adversely affected by the disease being treated.

An alternative strategy is to design treatment paradigms in which thedrug is dosed directly to the inflamed organ (topical therapy). Whilethis approach is not suitable for treating all chronic inflammatorydiseases, it has been extensively exploited in lung diseases (asthma,COPD), skin conditions (atopic dermatitis and psoriasis), nasal diseases(allergic rhinitis) and gastrointestinal disorders (ulcerative colitis).

In topical therapy, efficacy can be achieved either by ensuring that thedrug has a sustained duration of action and is retained in the relevantorgan to minimize the risks of systemic toxicity or by producing aformulation which generates a “reservoir” of the active drug. which isavailable to sustain its desired effects. The first approach isexemplified by the anticholinergic drug tiotropium (Spiriva). Thiscompound is administered topically to the lung as a treatment for COPD,and has an exceptionally high affinity for its target receptor,resulting in a very slow off rate and a consequent sustained duration ofaction.

In one aspect of the disclosure the compound of formula (I) isparticularly suitable for topical delivery, such as topical delivery tothe lungs, in particular for the treatment of respiratory disease, forexample chronic respiratory diseases such as COPD and/or asthma.

In one embodiment the compound of formula (I) is suitable forsensitizing patients to treatment with a corticosteroid who have becomerefractory to such treatment regimens.

The compound of formula (I) may also be useful for the treatment ofrheumatoid arthritis.

The compound of formula (I) may have antiviral properties, for examplethe ability to prevent infection of cells (such as respiratoryepithelial cells) with a picornavirus, in particular a rhinovirus,influenza or respiratory syncytial virus.

Thus the compound is thought to be an antiviral agent, in particularsuitable for the prevention, treatment or amelioration of picornavirusinfections, such as rhinovirus infection, influenza or respiratorysyncytial virus.

In one embodiment the compound of formula (I) is able to reduceinflammation induced by viral infection, such as rhinovirus infectionand in particular viral infections that result in the release ofcytokines such as IL-8, especially in vivo. This activity may, forexample, be tested in vitro employing a rhinovirus induced IL-8 assay asdescribed in the Examples herein.

In one embodiment the compound of formula (I) is able to reduce ICAM1expression induced by rhinovirus, especially in vivo. ICAM1 is thereceptor mechanism used by so-called major groove rhinovirus serotypesto infect cells. This activity may be measured, for example by a methoddescribed in the Examples herein.

It is expected that the above properties render the compound of formula(I) particularly suitable for use in the treatment and/or prophylaxis ofexacerbations of inflamatory diseases, in particular viralexacerbations, in patients with one or more of the following chronicconditions such as congestive heart failure, COPD, asthma, diabetes,cancer and/or in immunosuppressed patients, for example post-organtransplant.

In particular, the compound of formula (I) may be useful in thetreatment of one or more respiratory disorders including COPD (includingchronic bronchitis and emphysema), asthma, paediatric asthma, cysticfibrosis, sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis,rhinitis, sinusitis, especially asthma, and COPD (including chronicbronchitis and emphysema).

The compound of formula (I) may also be useful in the treatment of oneor more conditions which may be treated by topical or local therapyincluding allergic conjunctivitis, conjunctivitis, allergic dermatitis,contact dermatitis, psoriasis, ulcerative colitis, inflamed jointssecondary to rheumatoid arthritis or to osteoarthritis.

It is also expected that the compound of formula (I) may be useful inthe treatment of certain other conditions including rheumatoidarthritis, pancreatitis, cachexia, inhibition of the growth andmetastasis of tumours including non-small cell lung carcinoma, breastcarcinoma, gastric carcinoma, colorectal carcinomas and malignantmelanoma.

The compound of formula (I) may be useful in the treatment of eyediseases or disorders including allergic conjunctivitis, conjunctivitis,diabetic retinopathy, macular oedema (including wet macular oedema anddry macular oedema), post-operative cataract inflammation or,particularly, uveitis (including posterior, anterior and pan uveitis).

The compound of formula (I) may be useful in the treatment ofgastrointestinal diseases or disorders including ulcerative colitis orCrohn's disease.

The compound of formula (I) may also re-sensitise the patient'scondition to treatment with a corticosteroid, when the patient'scondition has become refractory to the same.

Furthermore, the present invention provides a pharmaceutical compositioncomprising a compound according to the disclosure optionally incombination with one or more pharmaceutically acceptable diluents orcarriers.

The present invention also provides a process for preparing such apharmaceutical composition which comprising mixing the ingredients.

Diluents and carriers may include those suitable for parenteral, oral,topical, mucosal and rectal administration.

As mentioned above, such compositions may be prepared e.g. forparenteral, subcutaneous, intramuscular, intravenous, intra-articular orperi-articular administration, particularly in the form of liquidsolutions or suspensions; for oral administration, particularly in theform of tablets or capsules; for topical e.g. pulmonary or intranasaladministration, particularly in the form of powders, nasal drops oraerosols and transdermal administration; for mucosal administration e.g.to buccal, sublingual or vaginal mucosa, and for rectal administratione.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985). Formulations for parenteral administration may contain asexcipients sterile water or saline, alkylene glycols such as propyleneglycol, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. Formulationsfor nasal administration may be solid and may contain excipients, forexample, lactose or dextran, or may be aqueous or oily solutions for usein the form of nasal drops or metered sprays. For buccal administrationtypical excipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or morephysiologically compatible carriers and/or excipients and may be insolid or liquid form. Tablets and capsules may be prepared with bindingagents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, orpoly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch,calcium phosphate, sorbitol, or glycine; lubricants, such as magnesiumstearate, talc, polyethylene glycol, or silica; and surfactants, such assodium lauryl sulfate. Liquid compositions may contain conventionaladditives such as suspending agents, for example sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or ediblefats; emulsifying agents such as lecithin, or acacia; vegetable oilssuch as almond oil, coconut oil, cod liver oil, or peanut oil;preservatives such as butylated hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT). Liquid compositions may be encapsulated in, forexample, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsulesand soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40% to 60% w/wconcentration of gelatin, about a 20% to 30% concentration ofplasticizer (such as glycerin, sorbitol or propylene glycol) and about a30% to 40% concentration of water. Other materials such aspreservatives, dyes, opacifiers and flavours also may be present. Theliquid fill material comprises a solid drug that has been dissolved,solubilized or dispersed (with suspending agents such as beeswax,hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug invehicles or combinations of vehicles such as mineral oil, vegetableoils, triglycerides, glycols, polyols and surface-active agents.

Suitably the compound of formula (I) is administered topically to thelung. Hence we provide according to the invention a pharmaceuticalcomposition comprising Compound (I) of the disclosure optionally incombination with one or more topically acceptable diluents or carriers.Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane (propellant 11),dichlorotetrafluoro methane (propellant 114), anddichlorodifluoromethane (propellant 12). Suitable HFC propellantsinclude tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).The propellant typically comprises 40% to 99.5% e.g. 40% to 90% byweight of the total inhalation composition. The formulation may compriseexcipients including co-solvents (e.g. ethanol) and surfactants (e.g.lecithin, sorbitan trioleate and the like). Aerosol formulations arepackaged in canisters and a suitable dose is delivered by means of ametering valve (e.g. as supplied by Bespak, Valois or 3M).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.This may be administered by means of a nebuliser. Nebulisers may beportable or non-portable Topical administration to the lung may also beachieved by use of a dry-powder formulation. A dry powder formulationwill contain the compound of the disclosure in finely divided form,typically with a mass mean aerodynamic diameter (MMAD) of 1-10 μm. Theformulation will typically contain a topically acceptable diluent suchas lactose, usually of larger particle size e.g. an MMAD of 50 μm ormore, e.g. 100 μm or more. An alternative topically acceptable diluentis mannitol. Examples of dry powder delivery systems include SPINHALER,DISKHALER, TURBOHALER, DISKUS and CLICKHALER. Further examples of drypowder inhaler systems include ECLIPSE, ROTAHALER, HANDIHALER,AEROLISER, CYCLOHALER, BREEZHALER/NEOHALER, FLOWCAPS, TWINCAPS, X-CAPS,TURBOSPIN, ELPENHALER, TURBUHALER, MIATHALER, TWISTHALER, NOVOLIZER,SKYEHALER, ORIEL dry powder inhaler, MICRODOSE, ACCUHALER, PULVINAL,EASYHALER, ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER, GYROHALER, TAPER,CONIX, XCELOVAIR and PROHALER.

One aspect of the invention relates to a dry powder pharmaceuticalformulation for inhalation comprising:

-   -   (i) Compound (I)

-   -   that is        1-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)-3-(4-(2-(phenylamino)pyrimidin-4-yloxy)naphthalen-1-yl)urea        or a pharmaceutically acceptable salt thereof, including all        stereoisomers and tautomers thereof, in particulate form (e.g.        solid crystalline Form B) as active ingredient;    -   (ii) particulate lactose as carrier; and    -   (iii) a particulate metal salt of stearic acid, such as        magnesium stearate.

The invention also provides for an inhalation device comprising one ormore doses of said formulation.

The compound of formula (I) has therapeutic activity. In a furtheraspect, the present invention provides a compound of the disclosure foruse as a medicament. Thus, in a further aspect, the present inventionprovides a compound as described herein for use in the treatment of oneor more of the above mentioned conditions.

In a further aspect, the present invention provides use of Compound (I)as described herein for the manufacture of a medicament for thetreatment of one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method oftreatment of one or more of the above mentioned conditions whichcomprises administering to a subject an effective amount of Compound (I)of the disclosure or a pharmaceutical composition comprising thecompound.

The word “treatment” is intended to embrace prophylaxis as well astherapeutic treatment.

Compound (I) of the invention may also be administered in combinationwith one or more other active ingredients e.g. active ingredientssuitable for treating the above mentioned conditions. For examplepossible combinations for treatment of respiratory disorders includecombinations with steroids (e.g. budesonide, beclomethasonedipropionate, fluticasone propionate, mometasone furoate, fluticasonefuroate), beta agonists (e.g. terbutaline, salbutamol, salmeterol,formoterol) and/or xanthines (e.g. theophylline). Other suitable activesinclude anticholinergics, such as tiotropium and anti-viral agents suchas, but not limited to, zanamivir or oseltamivir, for example as thephosphate. Other anti-viral agents include peramivir and laninamivir.Further possible combinations for treatment of respiratory disordersinclude combinations with steroids such as flunisolide, ciclesonide andtriamcinolone; beta agonists such bambuterol, levalbuterol, clenbuterol,fenoterol, broxaterol, indacaterol, reproterol, procaterol andvilanterol; muscarinic antagonists, (e.g. ipratropium, tiotropium,oxitropium, glycopyrronium, glycopyrrolate, aclidinium, trospium) andleukotriene antagonists (e.g. zafirlukast, pranlukast, zileuton,montelukast). It will be understood that any of the aforementionedactive ingredients may be employed in the form of a pharmaceuticallyacceptable salt.

In one embodiment the compound of formula (I) and the other activeingredient(s) are co-formulated in the same pharmaceutical formulation.In another embodiment the other active ingredient(s) are administered inone or more separate pharmaceutical formulations.

Hence, another aspect of the invention provides a combination productcomprising:

-   (A) a compound of the present invention (i.e. a compound of    formula (I) as defined above, or a pharmaceutically acceptable salt    thereof); and-   (B) another therapeutic agent,

wherein each of components (A) and (B) is formulated in admixture with apharmaceutically-acceptable diluent or carrier.

In this aspect of the invention, the combination product may be either asingle (combination) pharmaceutical formulation or a kit-of-parts.

Thus, this aspect of the invention encompasses a pharmaceuticalformulation including a compound of the present invention and anothertherapeutic agent, in admixture with a pharmaceutically acceptablediluent or carrier (which formulation is hereinafter referred to as a“combined preparation”).

It also encompasses a kit of parts comprising components:

-   (i) a pharmaceutical formulation including a compound of the present    invention in admixture with a pharmaceutically acceptable diluent or    carrier; and-   (ii) a pharmaceutical formulation including another therapeutic    agent, in admixture with a pharmaceutically-acceptable diluent or    carrier,

which components (i) and (ii) are each provided in a form that issuitable for administration in conjunction with the other.

Component (i) of the kit of parts is thus component (A) above inadmixture with a pharmaceutically acceptable diluent or carrier.Similarly, component (ii) is component (B) above in admixture with apharmaceutically acceptable diluent or carrier.

The other therapeutic agent (i.e. component (B) above) may be, forexample, any of the active ingredients mentioned above in connectionwith the treatment of respiratory disorders. The data reportedhereinbelow in relation to the antiviral properties of the compound offormula (I) provides evidence that other antiviral therapies incombination with a compound of formula (I) would be useful in thetreatment or prevention of virally-induced exacerbations (for examplerespiratory viral infections) suffered by patients with respiratorydisease such as COPD and/or asthma and/or one or more of the indicationslisted above. Thus, in one aspect there is provided the use of Compound(I) in combination with an anti-viral therapy such as, but not limitedto, zanamavir or oseltamivir (for example oseltamivir phosphate) in thetreatment or prevention of respiratory viral infections suffered bypatients with respiratory disease such as COPD and/or asthma.

The inventors also believe that other antiviral therapies in combinationwith Compound (I) would be useful in the treatment or prevention ofvirally induced exacerbations (for example respiratory viral infections)in patients with chronic conditions other than respiratory diseases, forexample conditions such as congestive heart failure, diabetes, cancer,or conditions suffered by immunosuppressed patients, for examplepost-organ transplant. Thus, in a further aspect there is provided theuse of a compound of the invention in combination with an anti-viraltherapy, such as, but not limited to, zanamavir or oseltamivir (forexample oseltamivir phosphate), in the treatment or prevention ofrespiratory viral infections in patients with chronic conditions such ascongestive heart failure, diabetes, cancer, or in conditions suffered byimmunosuppressed patients, for example post-organ transplant.

EXPERIMENTAL SECTION

Abbreviations used herein are defined below (Table 1). Any abbreviationsnot defined are intended to convey their generally accepted meaning.

TABLE 1 Abbreviations AcOH glacial acetic acid Aq aqueous ATPadenosine-5′-triphosphate BALF bronchoalveolae lavage fluid BEGMbronchial epithelial cell growth media br broad BSA bovine serum albuminCatCart ® catalytic cartridge CDI 1,1-carbonyl-diimidazole COPD chronicobstructive pulmonary disease CXCL1 chemokine (C—X—C motif) ligand 1 ddoublet DCM dichloromethane DMSO dimethyl sulfoxide DSC differentialscanning calorimetry d-U937 cells PMA differentiated U-937 cells DVSdynamic vapour sorption (ES⁺) electrospray ionization, positive mode Etethyl EtOAc ethyl acetate FCS foetal calf serum FRET fluorescenceresonance energy transfer GSK3α glycogen synthase kinase 3α HBEC primaryhuman bronchial epithelial cells hr hour(s) HRP horseradish peroxidiseHRV human rhinovirus ICAM-1 inter-cellular adhesion molecule 1 IRinfrared JNK c-Jun N-terminal kinase KC keratinocyte chemoattractant Kddissociation constant LPS Lipopolysaccharide (M + H)⁺ protonatedmolecular ion MAPK mitogen protein activated protein kinase MAPKAP-K2mitogen-activated protein kinase-activated protein kinase-2 Me methylMeCN acetonitrile MeOH methanol MHz megahertz min minute(s) MIP1αmacrophage inflammatory protein 1 alpha MMAD mass median aerodynamicdiameter MOI multiplicity of infection m.p. melting point MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide m/z:mass-to-charge ratio NMR nuclear magnetic resonance (spectroscopy) PBMCperipheral blood mononuclear cell PBS phosphate buffered saline Phphenyl PHA phytohaemagglutinin PMA phorbol myristate acetate pTSA4-methylbenzenesulfonic acid q quartet RT room temperature RP HPLCreverse phase high performance liquid chromatography RSV respiratorysyncytical virus s singlet sat saturated SCX solid supported cationexchange (resin) SDS sodium dodecyl sulphate S_(N)Ar nucleophilicaromatic substitution t triplet TCID₅₀ 50% tissue culture infectiousdose TGA thermogravimetric analysis THF tetrahydrofuran TNFα tumornecrosis factor alpha XRPD X-ray powder diffraction

General Procedures

All starting materials and solvents were obtained either from commercialsources or prepared according to the literature citation. Unlessotherwise stated all reactions were stirred. Organic solutions wereroutinely dried over anhydrous magnesium sulfate. Hydrogenations wereperformed on a Thales H-cube flow reactor under the conditions stated.Column chromatography was performed on pre-packed silica (230-400 mesh,40-63 μm) cartridges to using the amount indicated. SCX was purchasedfrom Supelco and treated with 1M hydrochloric acid prior to use. Unlessstated otherwise the reaction mixture to be purified was first dilutedwith MeOH and made acidic with a few drops of AcOH. This solution wasloaded directly onto the SCX and washed with MeOH. The desired materialwas then eluted by washing with 1% NH₃ in MeOH.

Preparative Reverse Phase High Performance Liquid Chromatography:

Agilent Scalar column C18, 5 μm (21.2×50 mm), flow rate 28 mL min⁻¹eluting with a H₂O-MeCN gradient containing 0.1% v/v formic acid over 10min using UV detection at 215 and 254 nm. Gradient information: 0.0-0.5min; 95% H₂O-5% MeCN; 0.5-7.0 min; ramped from 95% H₂O-5% MeCN to 5%H₂O-95% MeCN; 7.0-7.9 min; held at 5% H₂O-95% MeCN; 7.9-8.0 min;returned to 95% H₂O-5% MeCN; 8.0-10.0 min; held at 95% H₂O-5% MeCN.

Analytical Methods

Reverse Phase High Performance Liquid Chromatography:

(Method 1): Agilent Scalar column C18, 5 μm (4.6×50 mm) or WatersXBridge C18, 5 μm (4.6×50 mm) flow rate 2.5 mL min⁻¹ eluting with aH₂O-MeCN gradient containing either 0.1% v/v formic acid (Method 1acidic) or NH₃ (Method 1 basic) over 7 min employing UV detection at 215and 254 nm.

Gradient information: 0.0-0.1 min, 95% H₂O-5% MeCN; 0.1-5.0 min, rampedfrom 95% H₂O-5% MeCN to 5% H₂O-95% MeCN; 5.0-5.5 min, held at 5% H₂O-95%MeCN; 5.5-5.6 min, held at 5% H₂O-95% MeCN, flow rate increased to 3.5mL min⁻¹; 5.6-6.6 min, held at 5% H₂O-95% MeCN, flow rate 3.5 mL min⁻¹;6.6-6.75 min, returned to 95% H₂O-5% MeCN, flow rate 3.5 mL min⁻¹;6.75-6.9 min, held at 95% H₂O-5% MeCN, flow rate 3.5 mL·min⁻¹; 6.9-7.0min, held at 95% H₂O-5% MeCN, flow rate reduced to 2.5 mL min⁻¹.

Reverse Phase High Performance Liquid Chromatography:

(Method 2): Agilent Extend C18 column, 1.8 μm (4.6×30 mm) at 40° C.;flow rate 2.5-4.5 mL min⁻¹ eluting with a H₂O-MeCN gradient containing0.1% v/v formic acid over 4 min employing UV detection at 254 nm.Gradient information: 0-3.00 min, ramped from 95% H₂O-5% MeCN to 5%H₂O-95% MeCN; 3.00-3.01 min, held at 5% H₂O-95% MeCN, flow rateincreased to 4.5 mL min⁻¹; 3.01 3.50 min, held at 5% H₂O-95% MeCN;3.50-3.60 min, returned to 95% H₂O-5% MeCN, flow rate reduced to 3.50 mLmin⁻¹; 3.60-3.90 min, held at 95% H₂O-5% MeCN; 3.90-4.00 min, held at95% H₂O-5% MeCN, flow rate reduced to 2.5 mL min⁻¹.

¹H NMR Spectroscopy:

Spectra were acquired on a Bruker Avance III spectrometer at 400 MHzusing residual undeuterated solvent as reference.

Dynamic Vapour Sorption:

Plots were obtained using a Surface Measurement Systems dynamic vaporsorption model DVS-1 using about 10 mg of the sample. The weight changewas recorded with respect to atmospheric humidity at 25° C. and wasdetermined using the following parameters: drying: 60 min under drynitrogen; equilibrium: 60 min/step; data interval: 0.05% or 2.0 min. Therelative humidity [RH %] measurement points were as follows:

first set: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 90, 80, 70, 60,50, 40, 30, 20, 10, 5

second set: 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 90, 80, 70, 60, 50,40, 30, 20, 10, 5, 0.

X-Ray Powder Diffraction:

Patterns were obtained on a PANalytical (Philips) X'PertPRO MPDdiffractometer equipped with a Cu LFF X-ray tube (45 kV; 40 mA;Bragg-Brentano; spinner stage) and were acquired using Cu Kα radiationunder the following measurement conditions: scan mode: continuous; scanrange: 3 to 50° 28; step size: 0.02°/step; counting time: 30 sec/step;spinner revolution time: 1 sec; incident beam path: program. divergenceslit: 15 mm; Soller slit: 0.04 rad; beam mask: 15 mm; anti scatter slit:1°; beam knife: +; Diffracted beam path: long anti scatter shield: +;Soller slit: 0.04 rad; Ni filter: +; detector: X'Celerator. Samples wereprepared by spreading on a zero background sample holder.

Infrared Spectroscopy:

Micro attenuated total reflectance (microATR) was used and the samplewas analyzed using a suitable microATR accessory and the followingmeasurement conditions: apparatus: Thermo Nexus 670 FTIR spectrometer;number of scans: 32; resolution: 1 cm⁻¹; wavelength range: 4000 to 400cm⁻¹; detector: DTGS with KBr windows; beamsplitter: Ge on KBr; microATR accessory: Harrick Split Pea with Si crystal.

Differential Scanning Calorimetry:

Data were collected on a TA-Instruments Q1000 MTDSC equipped with RCScooling unit. Typically 3 mg of each compound, in a standard aluminiumTA-Instrument sample pan, was heated at 10° C./min from 25 C to 300° C.A nitrogen purge at 50 mL/min was maintained over the sample.

Thermogravimetric Analysis:

Data were collected on a TA-Instruments Q500 thermogravimeter Typically10 mg of each sample was transferred into a pre-weighed aluminium panand was heated at 20° C./min from ambient temperature to 300° C. or<80[(w/w) %] unless otherwise stated.

Chemical Stability by HPLC:

Analyses were carried out on a Waters Xbridge C₁₈ column (3.0×150 mm;3.5 μm) using the following operating conditions: column temperature:40° C.; sample temperature: 5° C.; flow rate: 0.45 mL/min; injectionvolume: 7 μL; UV detection at 260 nm; mobile phase composition comprisedof Phase A: 10 mM ammonium acetate+0.1%, v/v trifluoroacetic acid inwater and Phase B: acetonitrile, using the gradient defined by theparameters below (Table 2).

TABLE 2 Gradient Conditions for Chemical Stability Studies by HPLC. %Composition at Run Time (min) Eluent 0 20 25 26 32 Phase A 70 0 0 70 70PhaseB 30 100 100 30 30

Experimental Methods for Biological Testing

Enzyme Inhibition Assays

The kinase enzyme binding activities of compounds disclosed herein weredetermined using a proprietary assay which measures active site-directedcompetition binding to an immobilized ligand (Fabian, M. A. et al.,Nature Biotechnol., 2005, 23:329-336). These assays were conducted byDiscoverX (formerly Ambit; San Diego, Calif.). The Kd value(Dissociation constant value) was calculated as the index of affinity ofthe compounds to each kinase.

Enzyme Inhibition Assays

The enzyme inhibitory activities of compounds disclosed herein weredetermined by FRET using synthetic peptides labelled with both donor andacceptor fluorophores (Z-LYTE, Invitrogen Ltd., Paisley, UK).

p38 MAPKα Enzyme Inhibition

The inhibitory activities of test compounds against the p38 MAPKαisoform (MAPK14: Invitrogen), were evaluated indirectly by determiningthe level of activation/phosphorylation of the down-stream molecule,MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 μL) was mixed with thetest compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL or 0.004μg/mL) for 2 hr at RT. The mix solution (2.5 μL) of the p38a inactivetarget MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 μM; aphosphorylation target for MAPKAP-K2) was then added and the kinasereaction was initiated by adding ATP (40 μM, 2.5 μL). The mixture wasincubated for 1 hr at RT. Development reagent (protease, 5 μL) was addedfor 1 hr prior to detection in a fluorescence microplate reader(Varioskan® Flash, ThermoFisher Scientific).

p38 MAPKγ Enzyme Inhibition

The inhibitory activities of compounds of the invention against p38MAPKγ(MAPK12: Invitrogen), were evaluated in a similar fashion to thatdescribed hereinabove. The enzyme (800 ng/mL, 2.5 μL) was incubated withthe test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), andappropriate ATP solution (2.5 μL, 400 μM) was then added to theenzymes/compound mixtures and incubated for 1 hr. Development reagent(protease, 5 μL) was added for 1 hr prior to detection in a fluorescencemicroplate reader (Varioskan® Flash, Thermo Scientific).

c-Src and Syk Enzyme Inhibition

The inhibitory activities of compounds of the invention against c-Srcand Syk enzymes (Invitrogen), were evaluated in a similar fashion tothat described hereinabove. The relevant enzyme (3000 ng/mL or 2000ng/mL respectively, 2.5 μL) was incubated with the test compound (either4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr atRT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5μL, 800 μM for c-Src, and 60 μM ATP for Syk) were then added to theenzyme/compound mixtures and incubated for 1 hr. Development reagent(protease, 5 μL) was added for 1 hr prior to detection in a fluorescencemicroplate reader (Varioskan® Flash, ThermoFisher Scientific).

GSK 3α Enzyme Inhibition

The inhibitory activities of test compounds against the GSK 3α enzymeisoform (Invitrogen), were evaluated by determining the level ofactivation/phosphorylation of the target peptide. The GSK3-α protein(500 ng/mL, 2.5 μL) was mixed with the test compound (2.5 μL at either 4μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRETpeptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, andATP (40 μM, 2.5 μL) were then added to the enzyme/compound mixture andthe resulting mixture incubated for 1 hr. Development reagent (protease,5 μL) was added for 1 hr prior to detection in a fluorescence microplatereader (Varioskan® Flash, ThermoFisher Scientific).

In all cases, the site-specific protease cleaves non-phosphorylatedpeptide only and eliminates the FRET signal. Phosphorylation levels ofeach reaction were calculated using the ratio of coumarin emission(donor) over fluorescein emission (acceptor), for which low ratiosindicate high phosphorylation and high ratios indicate lowphosphorylation levels. The percentage inhibition of each reaction wascalculated relative to non-inhibited control and the 50% inhibitoryconcentration (IC₅₀ value) was then calculated from theconcentration-response curve.

Cellular Assays

LPS-induced TNFα/IL-8 Release in d-U937Cells

U937 cells, a human monocytic cell line, were differentiated intomacrophage-type cells by incubation with PMA (100 ng/mL) for 48 to 72hr. Cells were pre-incubated with final concentrations of test compoundfor 2 hr and were then stimulated with LPS (0.1 μg/mL; from E. Coli:0111:84, Sigma) for 4 hr. The supernatant was collected fordetermination of TNFα and IL-8 concentrations by sandwich ELISA(Duo-set, R&D systems). The inhibition of TNFα production was calculatedas a percentage of that achieved by 10 μg/mL of BIRB796 at eachconcentration of test compound by comparison against vehicle control.The relative 50% effective concentration (REC₅₀) was determined from theresultant concentration-response curve. The inhibition of IL-8production was calculated at each concentration of test compound bycomparison with vehicle control. The 50% inhibitory concentration (IC₅₀)was determined from the resultant concentration-response curve.

LPS-induced TNFα Release in THP-1 Cells

THP-1 cells, a human monocytic cell line, were stimulated with 3 μg/mLof LPS (from E. Coli; 0111:84, Sigma) for 4 hr and the supernatantcollected for determination of the TNFα concentration by sandwich ELISA(Duo-set, R&D systems). The inhibition of TNFα production was calculatedat each concentration by comparison with vehicle control. The 50%inhibitory concentration (IC₅₀) was determined from the resultantconcentration-response curve.

Poly I:C-Induced/CAM-1 Expression in BEAS2B Cells

Poly I:C was used in these studies as a simple, RNA virus mimic. PolyI:C-Oligofectamine mixture (1 μg/mL Poly I:C, ±2% Oligofectamine, 25 μL;Invivogen Ltd., San Diego, Calif., and Invitrogen, Carlsbad, Calif.,respectively) was transfected into BEAS2B cells (human bronchialepithelial cells, ATCC). Cells were pre-incubated with finalconcentrations of test compounds for 2 hr and the level of ICAM-1expression on the cell surface was determined by cell-based ELISA. At atime point 18 hr after poly I:C transfection, cells were fixed with 4%formaldehyde in PBS (100 μL) and then endogenous peroxidase was quenchedby the addition of washing buffer (100 μL, 0.05% Tween in PBS:PBS-Tween) containing 0.1% sodium azide and 1% hydrogen peroxide. Cellswere washed with wash-buffer (3×200 μL). and after blocking the wellswith 5% milk in PBS-Tween (100 μL) for 1 hr, the cells were incubatedwith anti-human ICAM-1 antibody (50 μL; Cell Signaling Technology,Danvers, Mass.) in 1% BSA PBS overnight at 4° C.

The cells were washed with PBS-Tween (3×200 μL) and incubated with thesecondary antibody (100 μL; HRP-conjugated anti-rabbit IgG, Dako Ltd.,Glostrup, Denmark). The cells were then incubated with of substrate (50μL) for 2-20 min, followed by the addition of stop solution (50 μL, 1NH₂SO₄). The ICAM-1 signal was detected by reading and reading theabsorbance at 450 nm against a reference wavelength of 655 nm using aspectrophotometer. The cells were then washed with PBS-Tween (3×200 μL)and total cell numbers in each well were determined by readingabsorbance at 595 nm after Crystal Violet staining (50 μL of a 2%solution in PBS) and elution by 1% SDS solution (100 μL) in distilledwater. The measured OD 450-655 readings were corrected for cell numberby dividing with the OD595 reading in each well. The inhibition ofICAM-1 expression was calculated at each concentration of test compoundby comparison with vehicle control. The 50% inhibitory concentration(IC₅₀) was determined from the resultant concentration-response curve.

Cell Mitosis Assay

Peripheral blood mononucleocytes (PBMCs) from healthy subjects wereseparated from whole blood (Quintiles, London, UK) using a densitygradient (Histopaque®-1077, Sigma-Aldrich, Poole, UK). The PBMCs (3million cells per sample) were subsequently treated with 2% PHA(Sigma-Aldrich, Poole, UK) for 48 hr, followed by a 20 hr exposure tovarying concentrations of test compounds. At 2 hr before collection,PBMCs were treated with demecolcine (0.1 μg/mL; Invitrogen, Paisley,UK,) to arrest cells in metaphase. To observe mitotic cells, PBMCs werepermeabilised and fixed by adding Intraprep (50 μL; Beckman Coulter,France), and stained with anti-phospho-histone 3 (0.26 ng/L; #9701; CellSignalling, Danvers, Mass.) and propidium iodide (1 mg/mL;Sigma-Aldrich, Poole, UK,) as previously described (Muehlbauer P. A. andSchuler M. J., Mutation Research, 2003, 537:117-130). Fluorescence wasobserved using an ATTUNE flow cytometer (Invitrogen, Paisley, UK),gating for lymphocytes. The percentage inhibition of mitosis wascalculated for each treatment relative to vehicle (0.5% DMSO) treatment.

Rhinovirus-Induced IL-8 Release and ICAM-1 Expression

Human rhinovirus RV16 was obtained from the American Type CultureCollection (Manassas, Va.). Viral stocks were generated by infectingHela cells with HRV until 80% of the cells were cytopathic.

BEAS2B cells were infected with HRV at an MOI of 5 and incubated for 2hr at 33° C. with gentle shaking for to promote absorption. The cellswere then washed with PBS, fresh media added and the cells wereincubated for a further 72 hr. The supernatant was collected for assayof IL-8 concentrations using a Duoset ELISA development kit (R&Dsystems, Minneapolis, Minn.).

The level of cell surface ICAM-1 expression was determined by cell-basedELISA. At 72 hr after infection, cells were fixed with 4% formaldehydein PBS. After quenching endogenous peroxidase by adding 0.1% sodiumazide and 1% hydrogen peroxide, wells were washed with wash-buffer(0.05% Tween in PBS: PBS-Tween). After blocking well with 5% milk inPBS-Tween for 1 hr, the cells were incubated with anti-human ICAM-1antibody in 5% BSA PBS-Tween (1:500) overnight. Wells were washed withPBS-Tween and incubated with the secondary antibody (HRP-conjugatedanti-rabbit IgG, Dako Ltd.). The ICAM-1 signal was detected by addingsubstrate and reading at 450 nm with a reference wavelength of 655 nmusing a spectrophotometer. The wells were then washed with PBS-Tween andtotal cell numbers in each well were determined by reading absorbance at595 nm after Crystal Violet staining and elution by 1% SDS solution. Themeasured OD₄₅₀₋₆₅₅ readings were corrected for cell number by dividingwith the OD595 reading in each well. Compounds were added 2 hr beforeHRV infection and 2 hr after infection when non-infected HRV was washedout.

Assessment of HRV16 Induced CPE in MRC5 Cells

MRC-5 cells were infected with HRV16 at an MOI of 1 in DMEM containing5% FCS and 1.5 mM MgCl₂, followed by incubation for 1 hr at 33° C. topromote adsorption. The supernatants were aspirated, and then freshmedia added followed by incubation for 4 days. Where appropriate, cellswere pre-incubated with compound or DMSO for 2 hr, and the compounds andDMSO added again after washout of the virus.

Supernatants were aspirated and incubated with methylene blue solution(100 μL, 2% formaldehyde, 10% methanol and 0.175% Methylene Blue) for 2hr at RT. After washing, 1% SDS in distilled water (100 μL) was added toeach well, and the plates were shaken lightly for 1-2 hr prior toreading the absorbance at 660 nm. The percentage inhibition for eachwell was calculated. The IC₅₀ value was calculated from theconcentration-response curve generated by the serial dilutions of thetest compounds.

In Vitro RSV Virus Load in Primary Bronchial Epithelial Cells.

Normal human bronchial epithelial cells (NHBEC) grown in 96 well plateswere infected with RSV A2 (Strain A2, HPA, Salisbury, UK) at an MOI of0.001 in the LHC8 Media:RPMI-1640 (50:50) containing 15 mM magnesiumchloride and incubated for 1 hr at 37° C. for adsorption. The cells werethen washed with PBS (3×200 μL), fresh media (200 μL) was added andincubation continued for 4 days. Where appropriate, cells werepre-incubated with the compound or DMSO for 2 hr, and then added againafter washout of the virus.

The cells were fixed with 4% formaldehyde in PBS solution (50 μL) for 20min, washed with washing buffer (3×200 μL; PBS including 0.5% BSA and0.05% Tween-20) and incubated with blocking solution (5% condensed milkin PBS) for 1 hr. Cells were then washed with washing buffer (3×200 μL)and incubated for 1 hr at RT with anti-RSV (2F7) F-fusion proteinantibody (40 μL; mouse monoclonal, lot 798760, Cat. No. ab43812, Abcam)in 5% BSA in PBS-tween). After washing, cells were incubated with anHRP-conjugated secondary antibody solution (50 μL) in 5% BSA inPBS-Tween (lot 00053170, Cat. No. P0447, Dako) and then TMB substrate(50 μL; substrate reagent pack, lot 269472, Cat. No. DY999, R&D Systems,Inc.) was added. This reaction was stopped by the addition of 2N H₂SO₄(50 μL) and the resultant signal was determined colorimetrically (OD:450 nm with a reference wavelength of 655 nm) in a microplate reader(Varioskan® Flash, ThermoFisher Scientific).

Cells were then washed and a 2.5% crystal violet solution (50 μL; lot8656, Cat. No. PL7000, Pro-Lab Diagnostics) was applied for 30 min.After washing with washing buffer, 1% SDS in distilled water (100 μL)was added to each well, and plates were shaken lightly on the shaker for1 hr prior to reading the absorbance at 595 nm. The measured OD₄₅₀₋₆₅₅readings were corrected to the cell number by dividing the OD450-655 bythe OD595 readings. The percentage inhibition for each well wascalculated and the IC₅₀ value was calculated from theconcentration-response curve generated from the serial dilutions ofcompound.

The Effect of Test Compounds on Cell Viability: MTT Assay

Differentiated 0937 cells were pre-incubated with each test compound(final concentration 1 μg/mL or 10 μg/mL in 200 μL media indicatedbelow) under two protocols: the first for 4 hr in 5% FCS RPM11640 mediaand the second in 10% FCS RPM11640 media for 24 h. The supernatant wasreplaced with new media (200 μL) and MTT stock solution (10 μL, 5 mg/mL)was added to each well. After incubation for 1 hr the media wereremoved, DMSO (200 μL) was added to each well and the plates were shakenlightly for 1 hr prior to reading the absorbance at 550 nm. Thepercentage loss of cell viability was calculated for each well relativeto vehicle (0.5% DMSO) treatment. Consequently an apparent increase incell viability for drug treatment relative to vehicle is tabulated as anegative percentage.

Cytokine Production in Sputum Macrophages from COPD.

Patients with COPD were inhaled with a nebulised solution of 3% (w/v)hypertonic saline using an ultrasonic nebuliser (Devilbiss, Carthage,Mo.) with tidal breathing for 5 min. This procedure was repeated amaximum of three times until enough sputum was obtained. The sputumsamples were homogenized and mixed vigorously using a vortex mixer in0.02% v/v dithiothreitol (DTT) solution. The samples were re-suspendedin PBS (40 mL) followed by centrifugation at 1500 rpm at 4° C. for 10min to obtain sputum cell pellets. The pellets were washed twice withPBS (40 mL). The sputum cells were then re-suspended in macrophageserum-free medium (macrophage-SFM, Life technologies, Paisley, UK; toachieve 2×10⁶/well in a 24 well plate) containing 20 U/mL penicillin,0.02 mg/mL streptomycin and 5 μg/mL amphotericin B and seeded on highbound 96-well plate, followed by incubation for 2 hr at 37° C. and at 5%CO₂ to allow the macrophages to attach to the bottom of the plate. Thecells on the plate were washed with fresh macrophage-SFM (200 μL/well)to remove neutrophils and other contaminated cells. The adherent cells(mainly sputum macrophages) on the plate were used for further analysis.Sputum induction and isolation were conducted in Quintiles Drug ResearchUnit at Guys Hospital and ethics approval and written informed consentwas obtained by Quintiles.

Where appropriate, 1 μL of a solution containing either the testcompound or reference article at the stated concentrations oralternatively 1 μL of DMSO as the vehicle control was added to each well(200 μL in media) and the cells were incubated for 2 hr. The cells werestimulated with LPS solution (50 μL, final concentration: 1 μg/mL) andincubated for 4 hr at 37° C. and 5% CO₂. The supernatant was thencollected and kept at −80° C. Millipore's luminex kits were used tomeasure the four analytes. After thawing the supernatant, the magneticantibody beads were multiplexed and incubated in a 96-well plate withstandard, background solution or the appropriate volume of sampleovernight with shaking at 4° C. After washing twice with 200 μL of washbuffer provided by the kit per well using a magnetic plate washer, thebeads were incubated for 1 hr at RT with 25 μL of the biotin conjugatedantibody solution provided by the kit with shaking. Streptavidinsolution was added for 30 min with shaking at RT. After washing with 200uL wash buffer per well, the beads were resuspended in sheath fluid (150μL) and analyzed immediately. The level of each analyte in thesupernatant was calculated using Xcel Fit software with a 4 or5-parameter equation using each standard curve. The inhibitions of eachcytokine production were calculated at each concentration by comparisonwith vehicle control. The IC50 values were determined fromconcentration-inhibition curves using XL-Fit (idbs, Guildford, UK)

Cytokine Production in Primary Bronchial Epithelial Cells from COPD.

Primary airway epithelial cells obtained from patients with COPD werepurchased from Asterand (Royston, UK), and maintained in bronchialepithelial cell growth media that was prepared by mixing together LHC8(Invitrogen) (500 mL), with LHC9 (Invitrogen) (500 mL) and 3 μL ofretinoic acid solution (5 mg/mL in neat DMSO. The media was removed byaspiration and fresh BEGM (200 μL) was added to each well. Whereappropriate, 1 μL of a solution of the test compound at the stateconcentrations or 1 μL of DMSO as the vehicle control was added and thecells were incubated for 2 hr. The cells were stimulated with TNFα (50μL; final concentration 50 ng/mL) and then incubated for 4 hr at 37° C.and 5% CO₂. The supernatant was then collected and kept at −20° C.

The levels of IL-6 and IL-8 were determined by ELISA using R&D Systems'Human IL-6 and IL-8 Duoset® Elisa Kits. The inhibition of IL-6 and IL-8production was calculated at each concentration by comparison withvehicle control. The 50% inhibitory concentrations (IC₅₀) weredetermined from the resultant concentration-response curves using XL-Fit(idbs, Guildford, UK).

In Vivo Screening: Pharmacodynamics and Anti-Inflammatory Activity

LPS-Induced Neutrophil Accumulation in Mice

Non-fasted Balb/c mice were dosed by the intra tracheal route witheither vehicle, or the test substance at the indicated times (within therange 2-8 hr) before stimulation of the inflammatory response byapplication of an LPS challenge. At T=0, mice were placed into anexposure chamber and exposed to LPS (7.0 mL, 0.5 mg/mL solution in PBS)for 30 min). After a further 8 hr the animals were anesthetized, theirtracheas cannulated and BALF extracted by infusing and then withdrawingfrom their lungs 1.0 mL of PBS via the tracheal catheter. Total anddifferential white cell counts in the BALF samples were measured using aNeubaur haemocytometer. Cytospin smears of the BALF samples wereprepared by centrifugation at 200 rpm for 5 min at RT and stained usinga DiffQuik stain system (Dade Behring). Cells were counted using oilimmersion microscopy. Data for neutrophil numbers in BAL are shown asmean±S.E.M. (standard error of the mean). The percentage inhibition ofneutrophil accumulation was calculated for each treatment relative tovehicle treatment.

Cigarette Smoke Model

A/J mice (males, 5 weeks old) were exposed to cigarette smoke (4%cigarette smoke, diluted with air) for 30 min/day for 11 days using aTobacco Smoke Inhalation Experiment System for small animals (ModelSIS-CS; Sibata Scientific Technology, Tokyo, Japan). Test substanceswere administered intra-nasally (35 μL of solution in 50% DMSO/PBS) oncedaily for 3 days after the final cigarette smoke exposure. At 12 hrafter the last dosing, each of the animals was anesthetized, the tracheacannulated and bronchoalveolar lavage fluid (BALF) was collected. Thenumbers of alveolar macrophages and neutrophils were determined by FACSanalysis (EPICS® ALTRA II, Beckman Coulter, Inc., Fullerton, Calif.,USA) using anti-mouse MOMA2 antibody (macrophage) or anti-mouse 7/4antibody (neutrophil). BALF was centrifuged and the supernatant wascollected. The level of keratinocyte chemoattractant (KC; CXCL1) in BALFwas quantitated using a Quentikine® mouse KC ELISA kit (R&D systems,Inc., Minneapolis, Minn., USA).

Example 1—Preparation of Compound (I)

The following intermediates used to prepare Compound (I) of theinvention have been previously described and were prepared using theprocedures contained in the references cited below (Table 3).

TABLE 3 Previously Described Intermediates. Intermediate Structure Name,LCMS Data and Reference A

3-tert-butyl-1-p-tolyl-1H-pyrazol-5-amine. R^(t) 2.46 min (Method 1basic); m/z 230 (M + H)⁺, (ES⁺). Cirillo, P.F. et al., WO 2000/43384, 27Jul 2000. B

4-((2-chloropyrimidin-4-yl)oxy)naphthalen-1-amine. R^(t) 1.80 min(Method 2); m/z 272/274 (M + H)⁺, (ES⁺). Cirillo, P.F. et al., WO2002/92576, 21 Nov 2000.

Intermediate C:4-((4-Aminonaphthalen-1-yl)oxy)-N-phenylpyrimidin-2-amine

To a nitrogen purged solution of mixture of Intermediate B (50.0 g, 184mmol) and aniline (42.0 mL, 460 mmol) in THF (200 mL) was added pTSA(17.5 g, 92.0 mmol) in a single portion. The reaction mixture was heatedto 70° C. for 1.5 hr during which time which a precipitate formed. Themixture was cooled to RT and diluted with THF (200 mL). The precipitatewas collected by filtration, washed with THF (2×100 mL) and thensuspended in a heterogeneous mixture of DCM (600 mL) and aq. NaOH (2M,200 mL) and stirred vigorously for 1 hr, during which time the suspendedsolids dissolved. The layers were separated and the aq layer wasextracted with DCM (200 mL). The DCM extracts were combined, dried andevaporated in vacuo. The residue was triturated with ether (150 mL) andthe resulting solid was washed with ether (2×50 mL) to affordIntermediate C as an off white solid (26 g, 43%); Rt 1.95 min (Method2); m/z 329 (M+H)⁺ (ES⁺).

Compound (I):1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(44(2-(phenylamino)pyrimidin-4-yl)oxy)naphthalen-1-yl)urea

A heterogeneous mixture of a solution of Na₂CO₃ (3.84 g, 36 mmol) inwater (42 mL) and Intermediate A (10.5 g, 45.7 mmol) in isopropylacetate (130 mL, 1.082 mol) was stirred vigorously at RT for 5 min andwas then treated with phenyl carbonochloridate (5.77 mL, 45.7 mmol).Stirring of the mixture was continued for a further 4 hr after which thelayers were separated. The organic phase was added to a solution ofIntermediate C (10.0 g, 30.5 mmol) and triethylamine (423 μL, 3.05 mmol)in isopropyl acetate (60 mL, 511 mmol). The reaction mixture was warmedto 48° C. for 1 hr, then diluted with isopropyl acetate (190 mL) andcooled to RT for a further 18 hr, during which time a precipitateformed. The precipitate was isolated by filtration, washed withisopropyl acetate and then dried in vacuo at 40° C. to afford the titlecompound, Compound (1) as a white solid (anhydrous free base,polymorphic form A) (16.5 g, 92%); Rt 2.74 min (Method 2); m/z 584(M+H)⁺ (ES⁺); ¹H NMR (400 MHz, DMSO-d₆) δ: 1.30 (9H, s), 2.41 (3H, s),6.43 (1H, s), 6.58 (1H, d), 6.78 (1H, t), 6.97 (2H, t), 7.28 (2H, br m),7.39 (2H, d), 7.40 (1H, d), 7.49 (2H, d), 7.56 (1H, m), 7.63 (1H, m),7.82 (1H, dd), 7.95 (1H, d), 8.10 (1H, d), 8.40 (1H, d), 8.77 (1H, s),9.16 (1H, br s), 9.50 (1H, br s).

Example 2—Summary of In Vitro and In Vivo Screening Results

The in vitro profile of Compound (I) disclosed herein, as determinedusing the protocols described above, are presented below (Tables 4a-f)in comparison with a structurally related Reference Compound which isN-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide, which has beenpreviously described as a potent anti-inflammatory agent with anti-viralactivity (Ito, K. et al., WO 2010/112936, PCT/GB2010/050575, 7 Oct. 2010and Ito, K. et al., WO 2010/067130, PCT/GB2009/051702, 17 Jun. 2010.

The compound of the present invention demonstrates a very similarinhibitory profile to the Reference Compound in the range of kinaseenzyme assays with the marked exception of the inhibitory activity ofCompound (I) against the enzyme GSK3α, which is very much weaker thanthe Reference Compound (Table 4a).

TABLE 4a p38 MAPK, c-Src, Syk and GSK3 α Enzyme Profile of Compound (I)Test IC₅₀ Values for Enzyme Inhibition (nM) Compound p38 MAPKα p38 MAPKγc-Src Syk GSK3α Compound (I) 60 3739 22 334 >17000 Reference 12 344 5 4245 Compound

The kinase binding profile of Compound (I) of the present invention wasalso compared with the Reference Compound against p38 MAPK, HCK, cSrc,Syk, and GSK3α/β. Compound (I) displayed a very different phenotype,demonstrating profound inhibition of binding versus p38MAPK, HCK, cSrcand Syk kinases, without significant effect against GSK3a (Table 4b).

TABLE 4b Comparison of the Enzyme Binding Profile of Compound (I) withthe Reference Compound. Kd value for kinase binding (nM) Test p38 1338Compound MAPKα MAPKγ HCK cSrc Syk GSK3α GSK3β Compound (I) 20 43 8 10 1420000 1200 Reference 1 5 5 4 9 180 24 compound

The compound of the present invention demonstrates a similar profile tothe Reference Compound in cellular assays that reveal anti-inflammatoryproperties against endotoxin mediated release of both TNFα and IL-8(Table 4c). The profiles of the compounds are also similar in cellularsystems measuring their effects on respiratory virus replication (HRVinduced ICAM1 and CPE expression and RSV stimulated expression ofF-protein) as well as virus-induced inflammation (HRV evoked release ofIL-8; Table 4d).

TABLE 4c Inhibition of LPS Induced TNFα and IL-8 Release and PolyICInduced ICAM-Expression for Compound (I) LPS Induced Release (nM) IL-8PolyIC/ Test TNFα IC₅₀ ICAM1 (nM) Compound IC₅₀ (THP1) REC₅₀ (dU937)(dU937) IC₅₀ (BEAS2B) Compound 3.4 2.3 2.2 10.2 (I) Reference 13 0.131.3 2.1 Compound

TABLE 4d The Effect of Compound (I) on HRV-16 Propagation (CPE) andInflammation (Expression of ICAM-1 and IL-8 Release) and on RSVPropagation (F-Protein Expression). IC₅₀ Values IC₅₀ Values (nM) for HRV(nM) for RSV Stimulated Release/Expression Stimulated Expression TestIL-8 ICAM1 CPE F-Protein Substance (BEAS2B) (BEAS2B) (MRC5) (HBEC)Compound (I) 0.036 0.023 17.1 15.4 Reference 0.065 0.37 4.7 22.0Compound

The compound of the present invention demonstrated higher efficacy inpro-inflammatory cytokine production in sputum macrophage and bronchialepithelial cells obtained from COPD patients, which were largelyinsensitive to fluticasone propionate, a corticosteroid. (Table 4e).

TABLE 4e The Effect of Compound (I) and Fluticasone propionate onpro-inflammatory cytokine release in sputum macrophages and bronchialepithelial cell from COPD patients. IC₅₀ values (nM) and/or E max (% inparentheses)¹ for Test Substance Indicated Cells Type Cytokine Compound(I) Fluticasone Propionate IL-6 43 (79) (26) Sputum IL-8 68 (64) (19)Macrophage {open oversize brace} TNFα 17 (86) (18) MIP1α 7.5 (89)  (20)Bronchial IL-6  1.7 (100) (38) {open oversize brace} Epithelial CellIL-8 0.85 (100)  (17) ¹E-max values (maximum inhibiton) were calculatedas the % inhibition obtained at 0.1 μg/mL

However, advantageously, Compound (I) shows markedly less activity inassay systems that measure its impact on cell viability and celldivision (mitosis) indicating that the compound is likely to possess asuperior therapeutic index over the Reference Compound (Table 4f).

TABLE 4f Effect of Compound (I) on Cellular Viability and Cell DivisionMTT Assay¹ Cell viability at time point indicated in Mitosis Assay Testd-U937 Cells % Inhibition at 5 μg/mL Substance 4 h 24 h in PBMC CellsCompound (I) −ve −ve 31.3 Reference −ve +ve 87.8 Compound ¹Cellviability screen: −ve and +ve indicate the value is below and aboverespectively, the no significant effect threshold defined as 30%inhibition at 1 μg/mL at the time point indicated.

Treatment of mice with Compound (I) was found to produce a dosedependent inhibition on LPS-induced neutrophil accumulation and a timecourse experiment revealed that the drug substance had a long durationof action (Table 5).

TABLE 5 The Effects of Treatment with Compound (I) on LPS-Induced AirwayNeutrophilia in Mice. Neutrophil numbers in BALF (×10⁵/mL) Compound (I)at pre-dose time indicated (% inhibition)¹ (mg/mL) 2 hr 8 hr 12 hrVehicle 18.9 ± 2.5 — — 0.05 15.6 ± 2.1 (18) — — 0.2  9.8 ± 1.6 (48) — —1.0  4.4 ± 0.89 (77) 9.9 ± 1.8 (48) 18.3 ± 2.3 (4) ¹N = 8 per group

The result of treatment with Compound (I) on macrophage and neutrophilaccumulation in BALF in the mouse cigarette smoke model was investigated(Table 6a). The cigarette smoke model used for this study is reported tobe a corticosteroid refractory system, (Medicherla S. et al., J.Pharmacol. Exp. Ther., 2008, 324(3):921-9.) and it was confirmed thatfluticasone propionate did not inhibit either neutrophil or macrophageaccumulation into airways at 1.75 μg/mouse (35 μL, bid, i.n.), the samedose that produced >80% inhibition of LPS-induced neutrophilaccumulation.

Treatment of mice with Compound (I) was found to produce adose-dependent inhibition on both macrophage and neutrophil accumulationin BALF induced by cigarette smoke.

TABLE 6a The Effects of Treatment with Compound (I) on Tobacco Smoke inMice. Cell numbers in Treatment BALF × 10⁴/mL (% inhibition) Compound(I) (μg/mL) Macrophage Neutrophil Vehicle + Air 4.3 ± 0.45   2.6 ±0.21   Vehicle + Tobacco Smoke 14.4 ± 0.33   13.7 ± 0.31   0.32 13.3 ±0.20 (11) 12.4 ± 0.32 (12) 1.6 11.6 ± 0.42 (28) 10.5 ± 0.06 (29) 8.010.1 ± 0.42 (43)  9.1 ± 0.28 (41) 40  7.9 ± 0.20 (64)  7.9 ± 0.34 (52)The data for cell numbers are shown as the mean ± SEM, N = 5

Treatment of mice with Compound (I) also inhibited cigarette smokeinduced CXCL1 (KC) production in BALF in a dose-dependent manner (Table6b).

TABLE 6b The Effects of Treatment with Compound (I) on CXCL1 (KC)release in BALF on Tobacco Smoke in Mice. Treatment CXCL1 in BALFCompound (I) (μg/mL) pg/mL (% inhibition) Vehicle + Air 8.2 ± 0.30  Vehicle + Tobacco Smoke 13.6 ± 1.69    0.32 13.6 ± 1.69 (0)  1.6 12.2 ±0.96 (26) 8.0 11.4 ± 0.15 (41) 40    9.5 ± 0.84 (76) The data for CXCLlevel are shown as the mean ± SEM, N = 5

In summary, these results suggest that the Compound (I) has similaranti-inflammatory properties to the Reference Compound disclosed aboveand, advantageously, is associated with a superior therapeutic index.

Example 3: Preparation of Compound (I) as the Anhydrous Free Base inSolid, Crystalline, Polymorphic Form B

Compound (I) (398 g, in polymorphic form A) was taken up in acetone(3.98 L) and the solution heated to 50° C. NORIT A SUPRA (19.9 g, anactivated carbon) and diatomaceous earth, flux-calcined (3.98 g; afilter agent) were then added and the mixture was heated to reflux (56°C.) for 15 min. The mixture was filtered and the resulting solid waswashed with acetone (100 mL). The combined filtrate and washing acetonewas warmed to reflux (56° C.), and 900 mL of solvent was removed viadistillation under atmospheric pressure at 56° C. The mixture was cooledto 50° C. and water (398 mL) was then added over a period of 1 hr whilstthe temperature was maintained at 50° C. After an additional 30 min at50° C. the heterogeneous mixture was cooled to 20° C. over 6h and thenstirred at 20° C. for 10 hr. The resulting product was filtered and thecake was washed with acetone (318 mL). The product was dried in vacuo at45° C. for 20 hr to produce Compound (I) as the anhydrous free base insolid, crystalline, polymorphic form B (240.9 g; 60.5% yield).

The above method may optionally be adapted to facilitate crystallizationwith seeding.

Example 3a: Preparation of Compound (I) as the Anhydrous Free Base inSolid, Crystalline, Polymorphic Form B Containing Reduced ResidualSolvent

Optionally, Compound (I) as the anhydrous free base in solid,crystalline, polymorphic form B, as prepared according to the proceduredescribed above (Example 3) or a similar method, may be re-slurried fromwater in order to reduce residual solvent as follows:

Compound (I) as the anhydrous free base in solid, crystalline,polymorphic form B (230 g, as prepared according to Example 3) wassuspended in deionized water (2.30 L) and was stirred at 20° C. for 4hr. The mixture was filtered and the product was washed with deionizedwater (2×115 mL) and was then dried at 45° C. in vacuo to produceCompound (I) as the anhydrous free base in solid, crystalline,polymorphic form B containing reduced residual solvent (227 g, 98.7%).

Example 4: Micronization of Compound (I) as the Anhydrous Free Base inSolid, Crystalline, Polymorphic Form B

Micronized crystalline polymorphic form B of Compound (I) as theanhydrous free base was prepared using a jet mill micronization device(1.5 bar using a manual feeder with an injector pressure of 1.5 bar)(manufactured by Hosokawa Alpine). The particle size distribution wasmeasured using laser diffraction (Malvern Mastersizer 2000S instrument).Particle size distributions may be represented using D₁₀, D₅₀ and D₉₀values. The D₅₀ median value of particle size distributions is definedas the particle size in microns that divides the distribution in half.The measurement derived from laser diffraction is more accuratelydescribed as a volume distribution, and consequently the D₅₀ valueobtained using this procedure is more meaningfully referred to as a Dv₅₀value (median for a volume distribution). As used herein Dv values referto particle size distributions measured using laser diffraction.Similarly, D₁₀ and D₉₀ values, used in the context of laser diffraction,are taken to mean Dv₁₀ and Dv₉₀ values and refer to the particle sizewhereby 10% of the distribution lies below the D₁₀ value, and 90% of thedistribution lies below the D₉₀ value, respectively. Micronizedcrystalline polymorphic form B of Compound (I) as the anhydrous freebase had the following particle size distribution: D₁₀ of 0.850 μm; D₅₀of 1.941 μm and D₉₀ of 4.563 μm.

Example 5: XRPD Analysis of Compound (I) as the Anhydrous Free Base inSolid, Crystalline, Polymorphic Forms A and B

XRPD analysis of Compound (I) as the anhydrous free base in solidcrystalline polymorphic forms A and B (polymorphic form B was micronizedfollowing the procedure of Example 4) was undertaken using the methoddescribed in General Procedures. The resulting diffraction patterns areshown in FIGS. 1 and 2 respectively. Both XRPD patterns showeddiffraction peaks without the presence of a halo, thereby indicatingthat both materials are crystalline. Peaks and their intensities arelisted below (Table 7a and Table 7b).

TABLE 7a Characteristic XRPD peaks and their intensities for Compound(I) as the anhydrous free base in solid, crystalline, form A XRPD Peaks2-Theta Values¹ Intensities 7.8 19.7 8.7 20.8 10.3 22.6 11.2 23.1 12.424.6 15.2 25.5 16.2 26.7 17.5 27.4 ¹Values are ± 0.2 degrees

TABLE 7b Characteristic XRPD peaks for Compound (I) as the anhydrousfree base in solid crystalline form B, post micronization. XRPD peaks2-Theta Values¹ Intensities 3.9 16.7 6.1 18.3 7.7 18.7 8.6 19.9 10.920.9 11.8 22.0 12.7 22.6 14.3 25.2 15.9 28.9 ¹Values are ± 0.2 degrees

Example 6: Melting Point Determination of Compound (I) as the AnhydrousFree Base in Solid, Crystalline, Polymorphic Forms A and B

The melting points of Compound (I) as the anhydrous free base in solidcrystalline polymorphic forms A and B (the latter post micronization)were obtained using differential scanning calorimetry (DSC), asdescribed in the General Procedures. Polymorphic form A melted at 191.6°C. and polymorphic form B melted at 214.0° C. From the DSC data it wascalculated that form B had a higher heat of fusion than form A. Sinceform B also has a higher melting point than form A, this indicates thatpolymorphic forms A and B are monotropic related, meaning that highermelting polymorphic form B will be more stable than lower meltingpolymorphic form A, at all temperatures. As such, it can be expectedthat polymorphic form B is thermodynamically more stable thanpolymorphic form A.

Example 7: Thermal Analysis of Compound (I) as the Anhydrous Free Basein Solid, Crystalline, Polymorphic Form B, Post Micronization

Thermal analysis of Compound (I) as the anhydrous free base incrystalline polymorphic form B (micronized) was undertaken using TGA,DVS, XRPD analysis, IR spectroscopy and DSC as described in GeneralProcedures. Where appropriate, a sample at ambient temperature andrelative humidity (reference sample/“0 days”) was compared with samplesstored at various temperatures and relative humidities (comparativesamples).

Thermogravimetric Analysis:

The reference sample (t=0) and the comparative samples that were exposedprior to analysis to different storage conditions, were heated at a rateof 20° C./min from RT to 300° C. The TGA curve of the reference sample(t=0) is illustrated in FIG. 3 and the results for all samples aresummarised below (Table 7). As can be seen from FIG. 3, a weight loss of0.6% was observed in the temperature range from RT to 180° C., which wasdue to solvent evaporation. The weight loss that occurred above 180° C.was due to evaporation and decomposition of the product. Comparing thisweight loss profile with those of the comparative samples in Table 7, nosignificant differences were observed.

Dynamic Vapour Sorption:

The DVS isotherm plot for the micronized reference sample is illustratedin FIG. 4 and the DVS change in mass plot for the micronized referencesample is illustrated in FIG. 5. During the initial drying step, noweight loss was registered and the product showed no hygroscopicbehavior. The product adsorbed up to 0.4% moisture depending on theatmospheric humidity. The product was found to dry out completely andremained in the same crystalline solid state (form B) during the test,as evidenced by the IR spectrum and XRPD pattern being substantially thesame before and after the DVS analysis.

XRPD Analysis and IR Spectroscopy.

The XPRD diffraction pattern of the reference sample (t=0) isillustrated in FIG. 2 and the IR trace is illustrated in FIG. 6. Thediffraction pattern and IR trace were compared with those of thecomparative samples (exposed to different storage conditions) and theresults are summarised in Table 7. The diffraction patterns and IRtraces were identical for all samples.

Differential Scanning Calorimetry.

The reference sample (t=0) and comparative samples, previously exposedto different storage conditions, were heated at a rate of 10° C./minfrom 25° C. to 300° C. The DSC curve of the reference sample isillustrated in FIG. 7 and the results for all samples are summarisedbelow (Table 8). From FIG. 7, it is evident that the reference samplemelted with decomposition at 214.0° C.

TABLE 8 Thermal analysis of Compound (I) as the anhydrous free base insolid crystalline polymorphic form B post micronization. Storage TGAAppear- Conditions <100° C. <180° C. XRD^(1,2) IR DSC³ ance T = zero 0.6Cryst. Cryst. 214 off-white Ref Ref 1 week/80° C. NT NT ~Ref ~Ref NToff-white 1 week/70° C./ NT NT ~Ref ~Ref NT off-white 75% RH 4 weeks/RT/0.8 0.5 ~Ref ~Ref 214 off-white <5% RH 4 weeks/RT/ 0.5 0.4 ~Ref ~Ref 214off-white 56% RH 4 weeks/RT/ 0.9 0.5 ~Ref ~Ref 214 off-white 75% RH 4weeks/50° C. 1.3 0.5 ~Ref ~Ref 214 off-white 4 weeks/40° C./ 0.8 0.3~Ref ~Ref 214 off-white 75% RH ¹Cryst.: crystalline; ²~Ref: patternidentical with reference sample, ³max (° C.); NT: Not tested in thisassay

In summary, it is evident that Compound (I) as the anhydrous free basein solid, crystalline, polymorphic form B has good physical stability.

Example 8: HPLC Analysis of Compound (I) as the Anhydrous Free Base inSolid, Crystalline, Polymorphic Form B Post Micronization

The chemical stability of Compound (I) as the anhydrous free base insolid, crystalline, polymorphic form B, following micronization, wasdetermined by comparing a sample maintained at ambient temperature andrelative humidity (reference sample) with samples stored at varioustemperatures and relative humidities as set out hereinabove (thecomparative samples, Table 8). The reference and comparative sampleswere then analyzed by HPLC using the method described in GeneralProcedures and by visual inspection. The results from this study (datasummarised in Table 9) reveal that Compound (I), prepared as theanhydrous free base in solid, crystalline, polymorphic form B ischemically stable although some sensitivity to light was observed.

TABLE 9 Chemical stability of Compound (I) as the anhydrous free base insolid, crystalline, polymorphic form B, post micronization. Sum ofimpurities Storage by HPLC (%)¹ Appearance¹ Conditions 1 week 4 weeks 1week 4 weeks T = zero 0.66 NT off-white NT 0.3 day, ICH light² 1.44 NToff-white NT 80° C. 0.73 NT off-white NT 70° C./75% RH 0.73 NT off-whiteNT 40° C./75% RH 0.66 0.68 off-white off-white 50° C. 0.69 0.66off-white off-white RT/<5% RH NT 0.67 NT off-white RT/56% RH NT 0.67 NToff-white RT/75% RH NT 0.67 NT off-white ¹NT in this assay system;²Stimulated daylight: light cabinet 700 W/m².

Example 9—Preparation of Pharmaceutical Formulations

An exemplary pharmaceutical formulation of the invention would consistof 0.4 wt. % of Compound (I) (as the anhydrous free base in solidcrystalline polymorphic form B), 98.6 wt. % lactose monohydrate(inhalation grade) and 1.0 wt. % magnesium stearate, wherein the wt. %of all components is based on the weight of the dry pharmaceuticalformulation.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

What is claimed is:
 1. A compound of formula (I)

or a pharmaceutically acceptable salt thereof, including allstereoisomers and tautomers thereof.
 2. A compound according to claim 1,as the free base.
 3. A compound according to claim 2, as the anhydrousfree base in solid crystalline form.
 4. A compound according to claim 3,wherein the compound of formula (I) as the anhydrous free base is insolid crystalline form having the X-ray powder diffraction patternsubstantially as shown in FIG. 1 (Form A).
 5. A compound according toclaim 3, wherein the compound of formula (I) as the anhydrous free baseis in solid crystalline form having an X-ray powder diffraction patterncontaining one, two, three, four, five, six or seven peaks selected from(±0.2) 10.3, 15.2, 17.5, 23.1, 24.6, 26.7 and 27.4 degrees 2-theta.
 6. Acompound according to claim 3, wherein the compound of formula (I) asthe anhydrous free base is in solid crystalline form having the X-raypowder diffraction pattern substantially as shown in FIG. 2 (Form B). 7.A compound according to claim 3, wherein the compound of formula (I) asthe anhydrous free base is in solid crystalline form having an X-raypowder diffraction pattern containing one, two, three, four, five, six,seven or all eight peaks selected from (±0.2) 3.9, 6.1, 11.8, 14.3,16.7, 18.3, 18.7 and 28.9 degrees 2-theta.
 8. A pharmaceuticalcomposition comprising a compound according to any of claims 1 to 7, incombination with one or more pharmaceutically acceptable diluents orcarriers.
 9. A compound of formula (I) according to any of claims 1 to 7for use as a medicament.
 10. A compound of formula (I) according to anyof claims 1 to 7 or a pharmaceutical composition according to claim 8for use in the treatment or prevention of exacerbations in patientswhich chronic respiratory disease, such as COPD (including chronicbronchitis and emphysema), asthma, paediatric asthma, cystic fibrosis,sarcoidosis, idiopathic pulmonary fibrosis.
 11. A compound of formula(I) according to any of claims 1 to 7 or a pharmaceutical compositionaccording to claim 8 for use in the treatment or prevention of acondition selected from: COPD (including chronic bronchitis andemphysema), asthma, paediatric asthma, cystic fibrosis, sarcoidosis,idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis, sinusitis,allergic conjunctivitis, conjunctivitis, allergic dermatitis, contactdermatitis, psoriasis, ulcerative colitis, inflamed joints secondary torheumatoid arthritis or osteoarthritis, rheumatoid arthritis,pancreatitis, cachexia, inhibition of the growth and metastasis oftumours including non-small cell lung carcinoma, breast carcinoma,gastric carcinoma, colorectal carcinomas and malignant melanoma.
 12. Useof a compound of formula (I) according to any of claims 1 to 7 or apharmaceutical composition according to claim 8 for the manufacture of amedicament for the treatment or prevention of a condition selected from:COPD (including chronic bronchitis and emphysema), asthma, paediatricasthma, cystic fibrosis, sarcoidosis, idiopathic pulmonary fibrosis,allergic rhinitis, rhinitis, sinusitis, pulmonary hypertension, allergicconjunctivitis, conjunctivitis, allergic dermatitis, contact dermatitis,psoriasis, ulcerative colitis, inflamed joints secondary to rheumatoidarthritis or osteoarthritis, rheumatoid arthritis, pancreatitis,cachexia, inhibition of the growth and metastasis of tumours includingnon-small cell lung carcinoma, breast carcinoma, gastric carcinoma,colorectal carcinomas and malignant melanoma.
 13. A method of treatmentof a condition selected from COPD (including chronic bronchitis andemphysema), asthma, paediatric asthma, cystic fibrosis, sarcoidosis,idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis, sinusitis,allergic conjunctivitis, conjunctivitis, allergic dermatitis, contactdermatitis, psoriasis, ulcerative colitis, inflamed joints secondary torheumatoid arthritis or osteoarthritis, rheumatoid arthritis,pancreatitis, cachexia, inhibition of the growth and metastasis oftumours including non-small cell lung carcinoma, breast carcinoma,gastric carcinoma, colorectal carcinomas and malignant melanoma whichcomprises administering to a subject an effective amount of a compoundof formula (I) according to any of claims 1 to 7 or a pharmaceuticalcomposition according to claim
 8. 14. A compound of formula (I)according to any of claims 1 to 7 or a pharmaceutical compositionaccording to claim 8 for use as in the treatment or prevention ofrespiratory viral infections in patients with chronic conditions such ascongestive heart failure, diabetes, cancer, or in immunosuppressedpatients, for example post-organ transplant.
 15. A compound of formula(I) according to any of claims 1 to 7 or a pharmaceutical compositionaccording to claim 8 in combination with anti-viral therapy such aszanamivir or oseltamivir (for example oseltamivir phosphate), for use inthe treatment or prevention of respiratory viral infections in patientswith chronic conditions such as congestive heart failure, diabetes,cancer, or in immunosuppressed patients, for example post-organtransplant.
 16. A combination product comprising: (A) a compound offormula (I) according to any of claims 1 to 7; and (B) anothertherapeutic agent; wherein each of components (A) and (B) is formulatedin admixture with a pharmaceutically-acceptable diluent or carrier;wherein said combination product may be either a single pharmaceuticalformulation or a kit-of-parts.